Project Summary/Abstract In the USA alone, over half of the 100,000 people that undergo anterior cruciate ligament reconstruction and up to 80 per cent of the 500,000 that have rotator cuff repairs yearly will experience repair failure, mostly due to the inability to regenerate a functioning enthesis. The structural and mechanical gradients of entheses are critical for normal function and regenerating these gradients is a major challenge. The present study?s goal is to develop a two- component system in which an electrospun template (component 1) will be used to deliver physicochemical cues to engineered adipose-derived stem cells (ASCs) (component 2) to generate a countergradient of growth factors that will guide the regeneration of the gradient structure of the enthesis. Ultimately, we envision this strategy as part of an integrated reconstruction system to be deployed via state of the art arthroscopic surgical procedures, providing a template to promote complete tendon/ligament regeneration. The work proposed in this study will delve into the basis for enthesis regeneration, the missing link identified as a cornerstone for further development of this integrated system. To achieve this goal, the following specific aims are proposed: Aim 1 is to develop and characterize electrospun templates with physicochemical cues to drive regeneration of a functional, biomimetic enthesis, component 1. Aim 2 is to generate engineered ASCs with synthetic gene regulatory networks constructed with clustered regularly interspaced short palindromic repeats (CRISPR)-based logical gates that respond to stimuli to create countergradients of growth factors and drive regeneration of a functional enthesis, component 2. The electrospun templates will be characterized using standard protocols for structural and mechanical properties, in vitro elution of cumate, derived blue light gradient, in vitro cytocompatibility and in vivo biocompatibility. The engineered ASCs will be generated and their growth factor expression in response to stimuli will be evaluated by renilla reporter and on-cell western assays, while their paracrine effect on directing wild-type ASC differentiation in gradient patterns will be evaluated using a transwell assay under a defined cumate and light environment without a template. Results from this project will inform future research on interfacial tissue engineering and further development of a complete tendon/ligament reconstruction system that will alleviate the burden of reconstruction surgery failure.